2-(r2-thio)-10-[3-(4-r1-piperazin-1-yl) propyl]-10h-phenothiazine for treating a beta-amyloidopathy or an alpha-synucleopathy, and method for the diagnosis or prediagnosis thereof

ABSTRACT

The invention relates to 2-(R 2 -thio)-10-[3-(4-R 1 -piperazin-1-yl)propyl]-10H-phenothiazine according to general formula I, for treating a β-amyloidopathy or an α-synucleinopathy accompanied by a cerebral protein deposit and a reduced activity of the cerebral ABCC1-transporter. The invention also relates to a method for the diagnosis or prediagnosis of a β-amyloidopathy or an α-synucleopathy accompanied by a cerebral protein deposit and a reduced activity of the cerebral ABCC1-transporter, or for determining the risk of a proband suffering from such an illness, the proband already having accumulated substances transported by the cerebral ABCC1 transporter.

The accumulation of proteins or protein fragments (peptides) in thebrain is a significant feature of age-dependent neurodegenerativediseases. In Alzheimer's dementia (Alzheimer's disease, AD) and cerebralβ-amyloidopathy (CAA) the aggregation of β-amyloid peptides (Aβ) is atrigger factor, the basic mechanism being unknown. The Aβ proteostasis,i.e. the equilibrium of production and degradation/removal by means ofreceptors or proteases is disturbed in AD and CAA. However, so farlittle attention has been paid to the removal of Aβ peptides by cellulartransporters (ABC transporters). In Parkinson's disease, the proteinα-synuclein accumulates, which inter alia regulates the dopamine releasein the substantia nigra. In Parkinson's disease α-synucleinopathy it isknown that ABC transporters play a crucial role for transport (Kortekaaset al., Ann Neural 2005, 57, 176-179). Here there are severalsubfamilies A-G which can alternatingly transport various substrates(metabolites, medicaments, peptides, proteins, ions) and are even ableto replace each other in the transport function (e.g. ABCB1 and ABCC1,Tao et al. Cancer Chemotherapy and Pharmacology, 64, 5, 961-969).

It has been shown by means of various genetically modified mouse modelsthat the ABC transporter (a common structural element of the ABCtransporter is an ATP-binding cassette and a transport pore) ABCC1 is animportant protein/peptide transporter, in particular Aβ transporter,which has extraordinary functional effects on the cerebral proteinaccumulation. ABCC1 is also an important α-synuclein transporter.

The investigations of the transporter activity are shown as an examplehereinafter for Aβ transport.

In order to determine the ABCC1 activity in vivo, in APP-expressing,transgenic mice, the ABCB1, ABCG2 or ABCC1 transporter was removedgenetically (knockout mice) in each case.

Here it was found that:

i) the quantity of Aβ in the mice lacking the ABCC1 transporter wasincreased by a factor of 12,

ii) loss of the ABCB1 transporter only results in a three-fold increaseand

iii) loss of ABCG2 has no Aβ-accumulating effect.

It was therefore the object of the present invention to providesubstances which suitably influence the ABCC1 transporter in order tothus be able to treat neurodegenerative diseases, in particularβ-amyloidopathies or α-synucleopathies. This object was solved by2-(R²-thio)-10-[3-(4-R¹-piperazin-1-yl)propyl]-10H-phenothiazinesaccording to claim 1. Further preferred embodiments are obtained fromthe dependent claims.

In other words, the object was solved by2-(R²-thio)-10-[3-(4-R¹-piperazin-1-yl)propyl]-10H-phenothiazinesaccording to the general formula I

wherein the residues

R¹ and R² are the same or different and each independently of oneanother are C₁-C₆ alkyl groups, which independently of one anotheroptionally comprise another substituent selected from alkyl, aryl, acyl(preferably acetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy,arylthio, alkylthio groups and halogen atoms, wherein the respectivealkyl groups optionally comprise at least one further halogen atom andthe residue

R³ is located at one of the positions 6-9 of the phenothiazine ringsystem and is a hydrogen atom or an alkyl, aryl, acyl (preferablyacetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio oralkylthio group or a halogen atom, wherein the respective alkyl groupsoptionally comprise at least one further halogen atom or an NR⁴R⁵ or OR⁶group, wherein R⁴, R⁵ and R⁶ are the same or different and eachindependently of one another are selected from hydrogen and C₁-C₃ alkylgroups and the residue

R⁷ is located at one of the positions 1, 2 or 4 of the phenothiazinering system and is a hydrogen atom or an alkyl, aryl, acyl (preferablyacetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio oralkylthio group or a halogen atom, wherein the respective alkyl groupsoptionally comprise at least one further halogen atom or an NR⁸R⁹ orOR¹⁰ group, wherein R⁸, R⁹ and R¹⁰ are the same or different and eachindependently of one another are selected from hydrogen and C₁-C₃ alkylgroup, for treating a β-amyloidopathy or an α-synucleinopathyaccompanied by a cerebral protein deposit.

Furthermore, both in the case of α-synucleinopathies and in the case ofβ-amyloidopathies, there is a need to identify or to diagnose orprediagnose these diseases.

It was also the object of the invention to provide a method with whichα-synucleinopathies and also β-amyloidopathies can be diagnosed orprediagnosed. This object is solved by a method according to claim 11.Preferred embodiments are obtained from the dependent claims. In otherwords the object is solved by a method for the diagnosis or prediagnosisof a β-amyloidopathy or α-synucleopathy or for determining the risk of aproband to develop such an illness, wherein the proband already takessubstances transported by the cerebral ABCC1 transporter, consisting ofthe following steps:

a) determining the quantity of ingested substance in body fluid samplesof the proband at a specific time point;

b) repeating the determination of step a) at at least one further latertime point;

c) comparing the quantities determined in step a) and b) with quantitieswhich had been defined as characteristic at the same time points forprobands who at the time of the sampling showed no clinical symptoms ofa β-amyloidopathy or an α-synucleopathy.

The fact that the proband already takes at least one substance which istransported via the cerebral ABCC1 transporter means that this substanceneeds not to be administered. On the contrary it is already present inthe body of the proband, for example, as a result of a drug treatment ofanother disease. The body fluid samples of the proband which are studiedare preferably samples from blood plasma, blood serum and/or cerebralspinal fluid.

The β-amyloidopathy is preferably an Alzheimer's dementia, theα-synucleinopathy is preferably Parkinson's disease. Optionally, theα-synucleinopathy can also be a dementia with Lewy bodies (DLB).Substances which are transported via the cerebral ABCC1 transporter arepreferably selected from antibiotics (e.g. difloxacin, grepafloxacin),virostatics/antiviral medicaments (e.g. saquinavir, ritonavir),anti-allergics/antihistamines (e.g. cimetidine), cardio-vascularmedicaments (e.g. verapamil), antidepressants (e.g. citalopram),antihyperuricemics (e.g. probenecid), cytostatics (e.g. methotrexate,etoposit, edatrexate, ZD1694), vitamins/vitaminanalogues (e.g.methotrexate, folic acid, L-leucovorin), antiphlogistics (e.g.indomethacin), anti-epileptics (e.g. valproic acid), hormones/hormonederivatives (e.g. 17β-estradiol), leukotrienes (e.g. LTC4), fluorescentsamples (e.g. calcein, Fluo-3, BCECF, SNARF), GSH-,sulphate-orglucuronide-coupled metabolites of natural substances(endogenously produced), toxinsor of medicaments (e.g.2,4-dinitrophenyl-SG, bimane-SG, N-ethylmaleimide-SG, doxorubicin-SG,thiotepa-SG, cyclophosphamide-SG, melphalan-SG, chlorambucil-SG,ethacrynic acid-SG, metolachlor-SG, atrazine-SG, sulforaphan-SG,aflatoxin B1-epoxide-SG, 4-nitroquinolin 1-oxide-SG, As(SG)3,etoposide-gluc, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL)-3β-0-gluc, SN-38-gluc, 4-methylumbelliferyl-β-d-gluc,6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)-benzothiazolsulfate(E3040S)-gluc, leukotriene C4, prostaglandin A2-SG, 15-deoxy-Δ-12,14prostaglandin J2-SG, hydroxynonenal-SG, 17β-estradiol-17-β-d-gluc,glucuronosylbilirubin, bis-lucuronosylbilirubin,hyodeoxycholate-6-α-gluc, estron-3-sulfate,dehydroepiandrosteronesulfate, sulfatolithocholate) (see also Deeley R Get al.: Substrate recognition and transport by multi drug resistanceprotein 1 (ABCC1), FEBS letters 2006, 580 (4), pp. 1103-1111.)

This indirect analysis of the transport activity of ABCC1 transporterscan be used for the diagnosis/prediagnosis of a corresponding disease.In probands who already take ABCC1-transportable substances by otherroutes, the profile of the active substance concentration in bodyfluids, preferably blood plasma, serum and/or cerebrospinal fluid can beinvestigated. A time-dependent measurement for probands in whom there isa reduced ABCC1 transport activity compared with healthy probands showsa delayed or shifted substance concentration curve (concentration cplotted over time t), i.e. the maximum of the curve varies with time.

When a shifted curve is obtained compared with the healthy case, this isan indication of a changed ABCC1 transport activity. This means thatsubstances such as Aβ or α-synuclein are transported less efficientlyand is therefore an indication of a corresponding disease.

Both the mouse model and the pharmacological influencing of the ABCC1show that this is an important cellular transmembrane transporter forthe Aβ protein and imply that the blood-brain barrier and the plexuschoroideus occupy a key position for the Aβ release from the brain. Itcould be shown that the selective pharmacological activation of theABCC1 transporter significantly reduces the cerebral loading with Aβ andcan thus be used therapeutically for the treatment of diseases withdisturbed brain proteostasis. Furthermore, the analysis of thetransporter activity of the ABCC1 transporter as described above can beused for the indirect or direct diagnosis/prediagnosis of acorresponding disease. Direct analysis would be possible via theadministration of substances which are transported via the ABCC1transporter, and the determination thereof. The indirect analysis hasalready been described further above.

Changes to export mechanisms which are related to ABC transporters cansubstantially influence the temporal aggregation profile of Aβ and otherbrain proteins. Consequently an influencing of the function of the ABCC1transporter has a positive effect on the risk of developingneurodegenerative diseases, particularly Alzheimer's disease. “Treatmentof neurodegenerative diseases” in this sense comprises the preventionand also the treatment of pre-existing diseases.

The role of the ABC transporter in the Aβ release was initially studiedin such a manner that it was demonstrated that ABCC1 is able totransport Aβ. For this purpose in vitro transwell assays withendothelial cells (endothelialcelltranswellassay, ECTA) of primarycultivated capillary endothelial cells from mouse brains (cell cultureapproach) were used.

Primary cultures of endothelial cells from brain capillaries ofABCB1-deficient, ABCC1-deficient (knock out) mice and control mice(C57Bl/6, FVB/N) were used to study the Aβ-specific transport activity.The transport of Aβ from the abluminal (brain) into the luminal (blood)compartment is impaired in ABCB1-deficient and ABCC1-deficientendothelial cells. The mean Aβ transport rate during the first six hoursafter administration of Aβ peptides (Aβ42) was 2.2 pg/min for thecontrol cells. In contrast to this, the ABCC1-deficient cells onlyreached half the transport capacity (1.0 pg/min). In the ABCB1-deficientcells the Aβ transport was almost non-existent (0.3 pg/min). Furtherinvestigations of capillary endothelial cells and cells from the plexuschoroideus revealed that the ABCB1 transporter is strongly expressed inbrain capillary endothelial cells whereas the endothelial ABCC1expression in brain capillaries is lower.

The relative significance of members of the ABC transporter family wasthen investigated in vivo using newly generated ABCtransporter-deficient Alzheimer mouse models. The genetically modifiedmice each exhibit a deficiency (knock out) at specific ABC transportersABCG2, ABCB1 or ABCC1.

The Aβ immunohistochemistry of brain sections showed:

i) significant increases in the cortical number and the size ofAβ-positive plaques in ABCC1-deficient mice compared to control mice(see FIGS. 1 and 2 a-c).

ii) ABCB1-deficient mice showed a smaller increase in the number andsize of Aβ-plaques than ABCC1-deficient mice.

iii) No significant difference could be determined between control miceand ABCG2-deficient mice (FIG. 2 a-c).

In order to determine the quantity of buffer-soluble Aβ (mostly monomersand smaller oligomers) and of guanidine-soluble Aβ (mostly fibrillar oraggregated material), enzyme-coupled immune adsorption tests(enzyme-linkedimmunoabsorbentassays, ELISAs) were used for Aβ.

In agreement with the morphological results from theimmunohistochemistry, the ABCC1-deficient mice showed a significantincrease in aggregated Aβ compared to the control mice at allmeasurement time points. The cerebral loading with Aβ was greatest at anage of 25 weeks. At this time point, the Aβ values (Aβ42) were 12 timeshigher than in the control mice. Buffer-soluble Aβ also increased withage but after 25 weeks, at the time of the highest plaque loading, thevalues of the soluble Aβ in the ABCC1-deficient group decreasedsubstantially.

Further investigations were carried out which provided further proof forthe relationship between the possibly lacking removal by ABCC1 and theaggregation of Aβ.

The transport kinetics of ABC transporters depend inter alia on specificprotein/peptide characteristics such as the specific charge. TheDutch-type variant of the amyloid precursor protein (Dutch mutant,APP_(dt)) which introduces an additional negative charge near theinterface of the α-secretase of the APP and thus results in a severecerebral amyloidangiopathy (CAA) influence the elimination of Aβ_(dt)via the blood-brain barrier. The Western blot analyses of braincapillaries and plexuschoroideus (CP) from control mice showed a strongexpression of ABCB1 in cerebral capillary endothelial cells (BC) and ofABCC1 in CP (FIG. 3 d). Since ABC transporters play an important role inthe elimination of Aβ, it was assumed that ABC-transporter-deficient (atthe blood-brain barrier and at the blood plexus choroideus barrier)APP_(dt)-transgenic mice exhibit an increased accumulation of Aβ_(dt) inmeningeal vessels. The degree of CAA in the ABC-deficient APP_(dt) micewas quantified at the age of 24 months. In agreement with theassumption, at least 51% of the vessels were severely impaired (>75% ofthe vessel wall loaded with Aβ) in the ABCC1-deficient animals comparedto 23% in the controls (FIG. 3 c).

On the basis of these results, it was investigated how far the contentof soluble Aβ in the brain could be reduced/influenced byactive-substance-mediated activation of ABC transporters. Mice withamyloid deposits were treated for 30 days with theanti-emeticthiethylperazine (Torecan®,2-(ethylthio)-10-[3-(4-methylpiperazin-1-yl)propyl]-10H-phenothiazine).3 mg/kg body weight was administered intramuscularly twice daily. Thepreventative treatment began before the mice exhibited senile plaques.ELISA measurements of the treated animals showed a reduction in thequantity of Aβ of at least 31% in the treated mice compared tovehicle-treated animals (vehicle=water) (FIG. 3 e). The results arereproduced graphically in FIG. 3.

The capacity to remove Aβ proved to be a key factor in the regulation ofthe intracerebral accumulation of Aβ.

Thiethylperazine (Torecan®) proved to be a particularly efficientactivator of the ABCC1 transporter. Other derivatives starting from thesame scaffold also showed good results in the activation of the ABCC1transporter. The corresponding derivatives are represented in thegeneral formula I

wherein the residues

R¹ and R² are the same or different and each independently of oneanother are C₁-C₆ alkyl groups, which independently of one anotheroptionally comprise another substituent selected from alkyl, aryl, acyl(preferably acetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy,arylthio, alkylthio groups and halogen atoms, wherein the respectivealkyl groups optionally comprise at least one further halogen atom andthe residue

R³ is located at one of the positions 6-9 of the phenothiazine ringsystem and is a hydrogen atom or an alkyl, aryl, acyl (preferablyacetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio,alkylthio group or a halogen atom, wherein the respective alkyl groupsoptionally comprise at least one further halogen atom or an NR⁴R⁵ or OR⁶group, wherein R⁴, R⁵ and R⁶ are the same or different and eachindependently of one another are selected from hydrogen and C₁-C₃ alkylgroups and the residue

R⁷ is located at one of the positions 1, 2 or 4 of the phenothiazinering system and is a hydrogen atom or an alkyl, aryl, acyl (preferablyacetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio,alkylthio group or a halogen atom, wherein the respective alkyl groupsoptionally comprise at least one further halogen atom or an NR⁸R⁹ orOR¹⁰ group, wherein R⁸, R⁹ and R¹⁰ are the same or different and eachindependently of one another are selected from hydrogen and C₁-C₃ alkylgroup

These derivatives are accordingly well suited to the treatment ofneurodegenerative diseases, in particular β-amyloidopathies orα-synucleinopathies where the treatment, as already mentioned, comprisesboth the prevention and the treatment of pre-existing diseases. Thehalogen atom/the halogen atoms are preferably selected from fluorine andchlorine. The acyl groups (—(C=0)-R) of the residues R^(1,2,3,7) arepreferably acetyl groups (—C(=0)CH₃). preferably the residues R¹ and R²are the same or different and each independently of one another are aC₁-C₆ alkyl group or a C₁-C₆ alkyl group (preferably C₁ alkyl)substituted with an acetyl group and the residues R³ and R⁷ are hydrogenor an acetyl group. In a preferred embodiment the residues R¹ and R² arethe same or different and each independently of one another are a C₁-C₃alkyl group. It is further preferred that the residues R³ and R⁷ arehydrogen. It is particularly preferred that the residue R¹ is a methylgroup, the residue R² is an ethyl group and the residues R³ and R⁷ arehydrogen (thiethylperazine, Torecan®). When used for the treatment ofneurodegenerative diseases, it has proved advantageous to add furtheractive substances, preferably 1-benzohydrylpiperazines, most preferably1-benzohydryl-4-cinnamyl piperazine (cinnarizine).

Various neurodegenerative diseases can be treated with the2-(R²-thio)-10-[3-(4-R¹-piperazin-1-yl)propyl]-10H-phenothiazinederivatives according to the invention or can be diagnosed by means ofthe indirect analysis described above. In a particularly preferredembodiment, the neurodegenerative disease is a β-amyloidopathy, inparticular Alzheimer's dementia (AD). Another embodiment relates to thecase that the neurodegenerative disease is an α-synucleinopathy, inparticular Parkinson's disease (PD). Both diseases, i.e. β-amyloidopathyand α-synucleinopathies are characterized by cerebral protein depositswhich can be treated by means of an activation of the ABCC1 transporteror can be diagnosed by means of its activity.

Other diseases which can also be treated by activation of the ABCC1transporter or which can be diagnosed by means of the ABCC1 transporteractivity are mentioned hereinafter. Another treatable disease is thusLewy body dementia (LBD). This is also characterized by cerebral proteinaggregation, i.e. is an α-synucleinopathy like Parkinson's disease.

Another embodiment relates to the case that the neurodegenerativedisease is Huntington's disease (HD). Another embodiment relates to thecase that the neurodegenerative disease is a prion disease, inparticular Creutzfeld-Jacob disease (CJD) orfatalfamilialinsomnia (FFI).Another embodiment relates to the case that the neurodegenerativedisease is a tauopathy, in particular cortico-basal degeneration (CBD),Steel-Richardson-Olszewski syndrome (PSP, progressivesupranuclearpalsy)or Pick's disease (PiD). Another embodiment relates to the case that theneurodegenerative disease frontotemporaldegeneration (FTLD), inparticular ubiquitin-positive degeneration, TDP43-positive degenerationor forubiquitin and TDP43-negative degenerations. Another embodimentrelates to the case that the neurodegenerative disease is anamyotrophiclateralsclerosis (ALS). Another embodiment relates to thecase that the neurodegenerative disease is a spinocerebellarataxia (SCA)or spasticparaparesis (SPG). Another embodiment relates to the case thatthe neurodegenerative/neuroimmunological disease is multiple sclerosis(MS) or an MS-related syndrome, in particular ADEM or Devic's syndrome.

DESCRIPTION OF THE FIGURES

In the figures

FIG. 1 a shows that the cortical density of neuritic plaques inABCC1-deficient mice (ABCCI ko) is increased by ˜75%;

FIG. 1 b,c shows that the mean plaque size is increased (+34%) as aresult of the larger number of plaques (+63%) having a size of more than700 μm² and a lower frequency of smaller plaques (−24%). Error bars,standard error (n≧3);

FIG. 1 d shows that the IHC staining in ABCG2-deficient (ABCG2ko),ABCB1-deficient (ABCBIko), ABCC1-deficient (ABCCIko) mice and in controlmice shows a higher surface density of Aβ in ABCC1-deficient animals.Typical plaques of the same size are shown in section, scaling barsrepresent 500 μm (overview) and 50 μm (section) (*p<0.05);

FIG. 2 a shows that the plaque density in the cortex (coverage) inspecific ABC-transporter knockout mice is increased. In particularABCC1-deficient (ABCCIko) mice show an increased Aβ-amyloid loading(light-grey bars, in each case on the outside right in the individualgroupings), w=week on the abscissa;

FIG. 2 b shows that the total plaque size in ABCC1-deficient (ABCCIko)and ABCB1-deficient (ABCBIko) mice at the age of 25 weeks is increased,w=week on the abscissa;

FIG. 2 c shows that the total increase in the plaque size is associatedwith fewer smaller plaques and more larger plaques (>700 μm²) whereasthe number of medium-size plaques remains at the same value, error bars,standard error (n 5), *p<0.05;

FIG. 3 shows that the deficiency of ABCC1 promotes the accumulation ofAβ and Aβ_(dt) and that the activation of ABCC1 (by administration ofTorecan) reduces the Aβ values; where

FIG. 3 a shows that at an age of 25 weeks ABCC1 deficiency leads to amarked increase (−12 times) in insoluble Aβ; and

FIG. 3 b shows that the quantity of buffer-soluble Aβ42 at an age of 25weeks is noticeably reduced compared with 22 weeks (−56%). This isprobably due to the deposition in insoluble deposits. At the same agethe area covered by Aβ deposits which is measured in theimmunohistochemistry is increased by 83% (error bars, standard error n5, p<0.05);

FIG. 3 c shows that 53% of the blood vessels are severely impaired byCAA (>75% of the vessel walls exhibit Aβ). This relates toABCC1-deficient mice (ABCC1ko) compared to 23% in the controls (n=3);

FIG. 3 d shows that the expression of ABCC1 can be seen predominantly inthe plexus choroideus (CP) whereas ABCD1 is principally expressed in thecapillaries of the brain (BP);

FIG. 3 e shows that the activation of ABCC1 by thiethylperazine(Torecan) lowers the Aβ values in mice (−28%), error bars, standarderror (n=4, *p<0.05).

EXAMPLES Animals

APP-transgenic mice (APP, APP_(dt)) were obtained from The JacksonLaboratory (Bar Harbor, USA) and the University of Tübingen (Tübingen,Germany). The NEP-deficient mice were obtained from the Riken BrainResearch Institute (Saitama, Japan). ABCG2-, ABCB1-, and ABCC1-deficientmice were obtained from Taconic-Farms (Denmark). All transgenic andknockout mouse lines were hybridised for at least 9 generations in thegenetic FVB-background. The mice were held in a 12 h/12 h light/darkcycle at 23° C. with free access to food and water.

Methods Tissue Preparation

For the tissue preparation the mice were killed by cervical dislocationand perfused transcardially with PBS (phosphate-buffered, physiologicalsaline solution). The brain was removed and one hemisphere was stored inbuffered 4% paraformaldehyde for paraffin embedding andimmunohistochemistry. The other hemisphere was shock-frozen in liquidnitrogen and stored at −80° C. for biochemical analyses.

ELISA

ELISA kits (TH40HS, TK42HS) from The Genetics Company (TGC, Schlieren,Switzerland) were used for the quantification of Aβ. Brain hemisphereswere homogenised using PreCellys24 (12 s, 6,500 rpm). After addingcarbonate buffer (pH 8.0) the homogenisates were mixed using PreCellys(5 s, 5,000 rpm) and centrifuged for 90 min at 4° C. and 24,000 g, inorder to separate insoluble from soluble Aβ species. The remainingsupernatant (buffer-soluble fraction) was mixed with 8M guanidinehydrochloride in a ratio of 1:1.6. To extract the aggregated Aβspecies,the pellet was dissolved in 8 volumes of 5M guanidine hydrochloride,agitated at room temperature for 3 h and centrifuged at 24,000 g for 20min at 4° C. The remaining supernatant formed the guanidine-solublefraction (GuaHCl). Protein contents of all the samples were measuredthree times, using a Nanodrop1000 spectrophotometer (ThermoFisherScientific, Wilmington, USA). The ELISAs were carried out according tothe manufacturer's instructions using suitable dilutions.

Western Blots

Tissue homogenisates were prepared for the Western Blots. The totalprotein concentrations of the extracts were determined using a BCA assay(Pierce, part of Thermo Fisher Scientific, Rockford, USA). Afterelectrophoresis of 10 μg total protein per trace, the proteins wereblotted onto PVDF membranes. After blocking in 5% dry milk inTBST-buffer (50 mMTris pH 7.4, 150 mMNaCl, 0.1% Tween20) for 1 h at roomtemperature, the blots were studied either on ABCB1 (1:500, D-11, SantaCruz), ABCC1 (1:200, Alexis Bio) or β-actin (1:20.000, Sigma) overnightat 4° C. Anti-mouse-HRP, anti-rat-HRP oranti-hare-HRP were used asdetection antibodies. An Amersham ECL Plus Detectionkit and aRoperCoolSnap HQ² camera were used for visualisation.

Immunohistochemistry (IHC)

Formalin-fixed brains were embedded in paraffin and cut into 4 μm thicksections. After removing the paraffin, the sections were further treatedwith a BondMax™ Autostainer (Menarini/Leica, Germany). Immunostainingwas initiated after blocking of endogenic peroxidase (5 min) andepitoperetrieval for 5 min using 95% formic acid (for antibody 6F3D,Dako, Germany) and 70% formic acid (for antibody 4G8, Millipore,Germany). Primary antibodies were routinely incubated at roomtemperature for 30 min with the following dilutions: 6F3D (1:100), 4G8(1:500). Primary antibodies were detected with the BondMax™ Bond PolymerRefinedetectionkit and according to the DAB R30 standard protocol. Thesections were completely digitised with a resolution of 230 nm using aMiraxDesk/MiraxMidi scanner and then analysed automatically using theAxioVision software package (Zeiss, Germany).

Assessment of the Severity of the CAA

Brain sections of APP_(dt) were stained with 4G8-antibody. At least twonon-consecutive sections were studied for CAA of the meningeal vesselsin a masked manner. All meningeal vessels were counted manually and theseverity of the CAA was categorised as follows:

Category I: not adversely affected

Category II: ≦25% of the periphery positively stained

Category III: ≦50% of the periphery positively stained

Category IV: ≦75% of the periphery positively stained

Category V: ≦100% of the periphery positively stained

The average number of vessels for each category was calculated relativeto the total number of identified vessels.

Endothelial Cell Transwell Assay (ECTA)

Endothelial cells of mouse brain capillaries were prepared as describedin Coisne et al. (Coisne, C. et al. Mouse syngenic in vitro blood-brainbarrier model: a new tool to examine inflammatory events in cerebralendothelium. Laboratory Investigation; 85, 734-746 (2005)). At least 3-4week old mice were beheaded and the brains removed. Following dissectionof the brain stem, the white matter and the meninges, the tissue washomogenised in two volumes of wash buffer B (WBB) (Hanksbufferedsaltsolution (HBBS), 10 mM HEPES, 0.1% BSA) using a 15 mlglassdouncer (Wheaton Industries, Millville, N.J.; USA). One volume of30% dextran solution was added to the homogenisate. This was centrifugedtwice at 3,000 g and 4° C. The pellet containing the vessels wasresuspended in WBB and large vessels were broken up manually by harshpipetting of the solution. Vacuum filtration through 60 μm membranes(SEFAR, Switzerland) was used to separate large vessels from thecapillaries. After combined treatment with collagenase/dispase (HBSS, 10mM HEPES, 0.15 g/ml TCLK, 10 μg/ml DNAse-I, 1 mg/ml collagenase/dispase(Roche) single cell suspension was achieved by further harsh pipettingof the solution. Endothelial cells were inserted into Matrigel-coatedTranswell inserts (0.4 μmpores, Greiner Bio-One, Germany) having adensity of 120,000 cells per insert and allowed to grow on a supportingglial culture.

Sulphur yellow was used to determine the paracellular flux during theassay. The culture medium of the abluminal compartment was replaced witha solution containing 10 ng Ass42 (1.6 nM final concentration). Samplesfrom the luminal compartment were then taken after 2 h, 6 h or 24 h andthe Aβ content was determined with ELISA (TK42-highsense, TGC,Switzerland). The transport rate was described in Coisne et al. (Coisne,C. et al. Mouse syngenic in vitro blood-brain barrier model: a new toolto examine inflammatory events in cerebral endothelium. LaboratoryInvestigation; 85, 734-746 (2005)).

ELISA Statistics

TheLillieforsgoodness-of-fit test (alpha=0.05) was applied to the ELISAdata and to the log-transformed ELISA data to distinguish between theassumption of normally distributed sample data and the assumption oflog-normally distributed sample data. Despite the small sample size, thenull hypothesis (H₀) was dismissed for both sets of data for 5 of 44samples. In agreement with the observation of predominantly positive(skew) and strictly positive sample data, the assumption of normallydistributed data was rejected. Mean confidence intervals were calculatedassuming a basic log-normal distribution. The Wilcoxon rank-sum test wasapplied to compare the ELISA data of the various mouse strains for eachtime point.

1. A 2-(R²-thio)-10-[3-(4-R¹-piperazin-1-yl)propyl]-10H-phenothiazineuseful for treating a β-amyloidopathy or an α-synucleinopathyaccompanied by a cerebral protein deposit and a reduced activity of thecerebral ABCC1-transporter, according to the general formula I

wherein the residues R¹ and R² are the same or different and eachindependently of one another is a C₁-C₆ alkyl group, which independentlyof one another optionally comprises another substituent selected fromthe group consisting of alkyl, aryl, acyl, amino, nitro, sulfonyl,hydroxyl, alkoxy, aryloxy, arylthio, and alkylthio groups and halogenatoms, wherein the respective alkyl groups optionally comprise at leastone further halogen atom and the residue R³ is located at one of thepositions 6-9 of the phenothiazine ring system and is a hydrogen atom oran alkyl, aryl, acyl, amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy,arylthio, alkylthio group or a halogen atom, wherein the respectivealkyl groups optionally comprise at least one further halogen atom or anNR⁴R⁵ or OR⁶ group, wherein R⁴, R⁵, and R⁶ are the same or different andeach independently of one another is selected from the group consistingof hydrogen and C₁-C₃ alkyl groups and the residue R⁷ is located at oneof the positions 1, 2, or 4 of the phenothiazine ring system and is ahydrogen atom or an alkyl, aryl, acyl, amino, nitro, sulfonyl, hydroxyl,alkoxy, aryloxy, arylthio, or alkylthio group or a halogen atom, whereinthe respective alkyl groups optionally comprise at least one furtherhalogen atom or an NR⁸R⁹ or OR¹⁰ group, wherein R⁸, R⁹, and R¹⁰ are thesame or different and each independently of one another is selected fromthe group consisting of hydrogen and C₁-C₃ alkyl groups.
 2. A2-(R²-thio)-10-[3-(4-R¹-piperazin-1-yl)propyl]-10H-phenothiazineaccording to claim 1, characterized in that wherein the halogen atom/thehalogen atoms are selected from the group consisting of fluorine andchlorine.
 3. A2-(R²-thio)-10-[3-(4-R¹-piperazin-1-yl)propyl]-10H-phenothiazineaccording to claim 1, wherein R¹ and R² are the same or different andeach independently of one another is a C₁-C₃ alkyl group.
 4. A2-(R²-thio)-10-[3-(4-R¹-piperazin-1-yl)propyl]-10H-phenothiazineaccording to claim 1, wherein the residues R³ and R⁷ are hydrogen.
 5. A2-(R²-thio)-10-[3-(4-R¹-piperazin-1-yl)propyl]-10H-phenothiazineaccording to claim 1, wherein the residue R¹ is a methyl group, theresidue R² is an ethyl group, and the residues R³ and R⁷ are hydrogen.6. A composition comprising a2-(R²-thio)-10-[3-(4-R¹-piperazin-1-yl)propyl]-10H-phenothiazineaccording to claim 1, further comprising a further active substance. 7.A composition according to claim 6, wherein a 1-benzohydrylpiperazine isa further active substance.
 8. A composition according to claim 7,wherein 1-benzohydryl-4-cinnamyl piperazine is the1-benzohydrylpiperazine.
 9. A2-(R²-thio)-10-[3-(4-R¹-piperazin-1-yl)propyl]-10H-phenothiazinesaccording to claim 1, wherein the β-amyloidopathy is Alzheimer'sdementia.
 10. A2-(R²-thio)-10-[3-(4-R¹-piperazin-1-yl)propyl]-10H-phenothiazinesaccording to claim 1, wherein the α-synucleinopathy is Parkinson'sdisease or Lewy body dementia.
 11. Method for the diagnosis orprediagnosis of a β-amyloidopathy or α-synucleopathy accompanied by acerebral protein deposit and a reduced activity of the cerebralABCC1-transporter, or for determining the risk of a proband to developsuch an illness, wherein the proband has already taken substancestransported by the cerebral ABCC1 transporter, comprising the followingsteps: a) determining a quantity of ingested substance in body fluidsamples of the proband at a specific time point; b) repeating thedetermination of step a) at at least one further later time point; c)comparing the quantities determined in step a) and step b) withquantities which had been defined as characteristic at the same timepoints for probands who at the time of the sampling showed no clinicalsymptoms of a β-amyloidopathy or an α-synucleopathy.
 12. Method for thediagnosis or prediagnosis of a β-amyloidopathy or α-synucleopathy or fordetermining the risk of a proband to develop such a disease according toclaim 11, wherein the body fluid samples of the proband are samples ofat least one of blood plasma, blood serum, and cerebrospinal fluid. 13.Method for the diagnosis or prediagnosis of a β-amyloidopathy orα-synucleopathy or for determining the risk of a proband to develop sucha disease according to claim 11, wherein the β-amyloidopathy is anAlzheimer's dementia.
 14. Method for the diagnosis or prediagnosis of aβ-amyloidopathy or α-synucleopathy or for determining the risk of aproband to develop such a disease according to claim 11, wherein theα-synucleopathy is Parkinson's disease or Lewy body dementia.
 15. Methodfor the diagnosis or prediagnosis of a β-amyloidopathy or anα-synucleopathy or for determining the risk of a proband to develop sucha disease according to claim 11, wherein the substances transported viathe cerebral ABCC1 transporter are selected from the group consisting ofantibiotics, virostatics/antiviral medicaments,anti-allergics/antihistamines, cardio-vascular medicaments,antidepressants, antihyperuricemics, cytostatics, vitamins/vitaminanalogues, antiphlogistics, anti-epileptics, hormones/hormonederivatives, leukotrienes, fluorescent samples, GSH-, sulfate, orglucuronide-coupled metabolites of natural substances (endogenouslyproduced), toxins, and medicaments.